EP2739733A2 - Compositions d'acide nucléique, procédés et kits pour l'appariement rapide d'agents d'affinité - Google Patents

Compositions d'acide nucléique, procédés et kits pour l'appariement rapide d'agents d'affinité

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Publication number
EP2739733A2
EP2739733A2 EP12819442.0A EP12819442A EP2739733A2 EP 2739733 A2 EP2739733 A2 EP 2739733A2 EP 12819442 A EP12819442 A EP 12819442A EP 2739733 A2 EP2739733 A2 EP 2739733A2
Authority
EP
European Patent Office
Prior art keywords
interest
nucleic acid
polypeptide
acid composition
promoter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP12819442.0A
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German (de)
English (en)
Other versions
EP2739733B1 (fr
EP2739733A4 (fr
Inventor
Andrew Hayhurst
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Texas Biomedical Research Institute
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Texas Biomedical Research Institute
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Publication of EP2739733A2 publication Critical patent/EP2739733A2/fr
Publication of EP2739733A4 publication Critical patent/EP2739733A4/fr
Application granted granted Critical
Publication of EP2739733B1 publication Critical patent/EP2739733B1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification

Definitions

  • the present inventions relate to the field of molecular biology tools and methods for diagnostic and therapeutic applications. Specifically, the inventions relate to the selection of peptides or proteins with desired properties, like specific binding affinity or greater stability or improved solubility, from vast numbers of variants. Further, the invention may be applied to the identification and characterization of any pair of affinity agent(s) including non-amino- acid motifs like synthetic aptamers and haptens, or other molecular configurations that are not derived from a nucleic acid-based system.
  • Phage technology involves engineering bacteriophages to generate proteins or peptides from a library of variants.
  • the protein or peptide of interest can either be produced as soluble molecules or be displayed as coat protein fusions on the surface of a phage or phagemid particle.
  • Some phage constructs e.g. pHEN vectors
  • a phage construct may produce the displayed form of the protein in a host cell expressing the suppressor tKNA supE and the soluble form of the protein in a non-supE host cell.
  • This two-host cell system has several logistical and molecular disadvantages. It would be beneficial to work within a single host cell, and use a biomolecular switch to change the expression profile of the desired protein from the displayed form to the soluble form or vice-versa.
  • Phage technology is a powerful tool to study protein interactions with target molecules and it is useful in identifying epitopes, mimotopes, and. other functional and accessible sites of target molecules.
  • the technology is also useful in vaccine design and engineered phages can serve as vaccine delivery units.
  • One of the most dominant applications of phage technology is to generate proteins or peptides, especially monoclonal antibodies, with affinity to molecules of interest. Once libraries have been mined for desired properties, like specific binding properties or greater stabilit profiles, the subsequent characterization of those binding agents or affinity agents becomes a time consuming and expensive process. Whereas therapeutic applications may rely on a single affinity agent, many diagnostic applications require two unique affinity reagents that bind the target molecule non-competitiveiy.
  • Some diagnostic applications may function using a single affinity agent as long as the antigen or molecule of interest is ofigomeric (i.e. dimer or higher multimer).
  • agents and methods that screen for and utilize a single affinity agent as both the captor and the tracer.
  • agents and methods that screen for and utilize unique affinity agents as captor and tracer If reagents and methods could, be developed to satisfy these needs, it would enable rapid pairing of affinity agents in any circumstance, leading to increased efficiencies in time and resources.
  • One embodiment of the invention is a nucleic acid composition that contains a promoter and an expression cassette encoding a polypeptide of interest, a biotin substrate site and. a display protein, the expression cassette having a termination codon.
  • the promoter is an inducible promoter.
  • the polypeptide of interest is obtained from an antibody library.
  • the polypeptide of interest is a single domain antibody.
  • the expression cassette encodes a linker between the polypeptide of interest and the biotin substrate site.
  • the termination codon is between the polypeptide of interest and the display protein.
  • the expression cassette further encodes a biotinylating agent.
  • Another embodiment of the invention is a nucleic acid composition that contains an inducible promoter, and an expression cassette encoding a polypeptide obtained from a single domain antibody library, a biotin substrate site, a display protein, and a biotinylating agent, the expression cassette having a termmation codon between the sequences for the polypeptide and the display protein.
  • Another embodiment of the invention is a cell that contains a first nucleic acid composition that contains a first promoter and an expression cassette encoding a polypeptide of interest, a biotin substrate site, and a display protein, the expression cassette having a termination codon; and a second nucleic acid composition that contains a second promoter and a gene encoding a suppressor tRNA that recognizes the termination codon in the first nucleic acid composition.
  • Another embodiment of the invention is a method of producing a cell that contains a first nucleic acid composition that contains a first promoter and an expression cassette encoding a polypeptide of interest, a biotin substrate site, and a display protein, the expression cassette having a termination codon; and a second nucleic acid composition that contains a second promoter and a gene encoding a suppressor tRNA that recognizes the termination codon in the first nucleic acid composition.
  • kits that contains a nucleic acid composition that contains a promoter and an expression cassette encoding a polypeptide of interest, a biotin substrate site, and a display protein, and the expression cassette having a termination codon.
  • the kit further contains a second nucleic acid composition
  • HOUSTON4I30063 -3- including a second promoter and a gene encoding a suppressor tRNA that recognizes the termination codon in the nucleic acid composition.
  • Another embodiment of the invention is a method of generating biotinylated polypeptides of interest, the method including (a) producing a cell containing a nucleic acid composition containing a promoter and an expression cassette encoding a polypeptide of interest, a biotin substrate site, and a display protein, the expression cassette having a termination codon; (b) incubating the cell under conditions sufficient for expression of the polypeptide of interest with the biotin substrate site; and (c) providing an agent capable of biotinylating the polypeptide of interest at the biotin substrate site.
  • Another embodiment of the invention is a method of producing a cell containing a nucleic acid composition comprising a promoter and an expression cassette encoding a polypeptide of interest, a biotin substrate site, and a display protein, and the expression cassette having a termination codon.
  • Another embodiment of the invention is a method of generating biotinylated polypeptides of interest, the method containing the following steps: (a) producing a cell containing a first nucleic acid composition including a first promoter and an expression cassette encoding a polypeptide of interest, a biotin substrate site, and a display protein, the expression cassette having a termination codon; and a second nucleic acid composition containing a second promoter and a gene encoding a suppressor tRNA that recognizes the termination codon in the first nucleic acid composition; (b) incubating the cell under conditions sufficient for protein expression from the first promoter in the first nucleic acid composition and the second promoter in the second nucleic acid composition, whereby the polypeptide of interest with the biotin substrate site and. the display protein is produced; and (c) providing an agent capable of biotinylating the polypeptide of interest at the biotin
  • HOUSTON4I30063 -4- substrate site thereby producing biotinylated polypeptides of interest with the display protein.
  • Another embodiment of the invention is a method of screening for a polypeptide of interest with affinity to a molecule of interest, the method containing the following steps: (a) providing an array containing biotin affinity agents: (b) producing biotinylated polypeptides including (i) producing a cell that contain a nucleic acid composition containing a promoter and an expression cassette encoding a polypeptide of interest, a biotin substrate site, and a display protein, the expression cassette having a termination codon; (ii) incubating the cell under conditions sufficient for expression of the polypeptide of interest with the biotin substrate site; and (iii) providing an agent capable of biotinylating the polypeptide of interest at the biotin substrate site; (c) adding a cellular fraction including the biotinylated polypeptides to the array; (d) adding the molecule of interest to bind to the biotinylated polypeptides; (e) adding the cellular fraction containing the biotin
  • Another embodiment of the invention is a method of screening for a polypeptide of interest with affinity to a molecule of interest, the method containing the following steps: (a) providing an array including biotin affinity agents; (b) producing biotinylated polypeptides including (i) producing a cell containing a first nucleic acid composition including a first promoter and an expression cassette encoding a polypeptide of interest, a biotin substrate site, and a display protein, the expression cassette having a termination codon, and a second nucleic acid composition containing a second, promoter and a gene encoding a suppressor tR A that recognizes the termination codon in the first nucleic acid, composition; (ii) incubating the cell under conditions sufficient for protein expression from the first promoter in the first nucleic acid composition and.
  • the second promoter in the second nucleic acid composition whereby the polypeptide of interest with the biotin substrate site and the display protein is produced; and (iii) providing an agent capable of biotinylating the polypeptide of interest at the biotin substrate site, thereby producing biotinylated polypeptides of interest with the display protein; (c) adding a cellular fraction containing the biotinylated polypeptides to the array; (d) adding the molecule of interest to bind to the biotinylated polypeptides; (e) adding the cellular fraction including the biotinylated polypeptides; and (f) detecting the polypeptide of interest that exhibits binding affinity to the molecule of interest.
  • An embodiment of the invention is a cell that contains a first nucleic acid composition that contains a first promoter and a first expression cassette encoding a polypeptide of interest and a biotin substrate site; and a second nucleic acid composition that includes a second promoter and a second expression cassette encoding a biotin affinity agent and a display protein.
  • the cell further contains a termination codon between the biotin affinity agent and the display protem in the second nucleic acid composition and a nucleic acid composition for a suppressor tRNA that recognizes the termination codon.
  • Another embodiment of the invention is a method of generating biotinylated polypeptides of interest, the method containing the following steps: (a) producing a cell containing a first nucleic acid composition that has a first promoter and an expression cassette encoding a polypeptide of interest and a biotin substrate site; and a second nucleic acid composition that has a second promoter and a second expression cassette encoding a biotin affinity agent and a display protein; (b) incubating the cell under conditions sufficient for protein expression from the first promoter in the first nucleic acid composition and the second promoter in the second nucleic acid composition, whereby the polypeptide of interest with the biotin substrate site and the display protein with the biotin affinity agent are produced; and (c) providing an agent capable of biotinylating the polypeptide of interest at the biotin substrate site, thereby producing biotinylated polypeptides of interest and the display protein with the biotin affinity agent.
  • Another embodiment of the invention is a method of producing a cell containing a first nucleic acid composition that has a first promoter and an expression cassette encoding a polypeptide of interest and a biotin substrate site; and a second nucleic acid composition that has a second promoter and a second expression cassette encoding a biotin affinity agent and a display protein.
  • Another embodiment of the invention is a method of screening for a polypeptide of interest with affinity to a molecule of interest, the method containing the following steps: (a) providing an array including biotm affinity agents; (b) producing biotinylated polypeptides of interest including (i) producing a cell containing a first nucleic acid composition including a first promoter and an expression cassette encoding a polypeptide of interest and a biotin substrate site; and a second nucleic acid composition has a second promoter and a second expression cassette encoding a biotin affinity agent and a display protein; (ii) incubating the cell under conditions sufficient for protein expression from the first promoter in the first nucleic acid composition and the second promoter in the second nucleic acid composition, whereby the polypeptide of interest with the biotin substrate site and the display protein with the biotin affinity agent are produced; and (iii) providing an agent capable of biotmylating the polypeptide of interest at the biotin substrate site,
  • FIG. 1A is an illustration of the rapid affinity agent pairing method.
  • FIG. IB is an illustration of the nucleic acid compositions used to establish the rapid pairing system.
  • FIG. 1C is a graph that shows the rapid affinity agent pairing system using the biotinyiated antibodies as captors and phage displayed antibodies as tracers.
  • the x axis indicates the volume of shockate used and the y axis indicates the absorbance units from measuring the product of the horseradish peroxidase (HRP) activity.
  • the HRP here is conjugated to an anti-M13 monoclonal antibody,
  • FIG. ID is a graph that shows the rapid affinity agent pairing system using the biotinyiated antibodies as both captors and tracers.
  • the x axis indicates the volume of shockate used, and the y axis indicates the absorbance units from measuring the product of the horseradish peroxidase activity.
  • the HRP here is conjugated to neutravidin.
  • FIG. IE is a graph showing that recombinant Marburg and Ebola Zaire virus nucleoprotein mimics are acceptable surrogates for developing sdAbs against the respective viruses.
  • the x axis indicates the volume of crude E.coli lysate expressing the recombinant nucleoprotein, and the y axis indicates a measure of luminescence derived from the enzymatic activity of the alkaline phosphatase fusion to the sdAb.
  • FIG. IF is a graph that shows the effect of the birA mutations on the positive phage production levels, following the expression of various vectors in XL-1 Blue host and analyzed by a phage display ELISA.
  • the x axis indicates the dilution of supernatant containing the phage, and the y axis indicates the absorbance units from measuring the product of the horseradish peroxidase activity.
  • the HRP here is conjugated to an anti-M 13 monoclonal antibody.
  • FIG. 1G is a graph that shows the effect of the birA mutations on the positive phage production levels, following the expression of various vectors in HBV88 host and analyzed by a phage display ELISA.
  • the x axis indicates the dilution of supernatant containing the phage, and the y axis indicates the absorbance units measuring the product of the horseradish peroxidase activity.
  • the HRP here is conjugated to an anti-M13 monoclonal antibody.
  • FIG. 1H is a graph that shows the effect of the variations in linkers, between the BAP sequence and the sdAb/ His6 tag, on the positive phage production levels, following the expression of various vectors in XL-i Blue host and analyzed by a phage display ELISA.
  • the x axis indicates the dilution of supernatant containing the phage, and the y axis indicates the absorbance units measuring the product of the horseradish peroxidase activity.
  • the HRP here is conjugated to an anti-M13 monoclonal antibody,
  • FIG. II is a graph that shows the effect of the variations in linkers, between the BAP sequence and the sdAb/ His6 tag, on the positive phage production levels, following the rescue of various vectors in the HBV88 host in display mode and analyzed by a phage display ELISA .
  • the x axis indicates the dilution of supernatant containing the phage, and the y axis mdicates the absorbance units measuring the product of the horseradish peroxidase activity.
  • the HRP here is conjugated to an a ti-M13 monoclonal antibody.
  • FIG. 1J is a schematic drawing of the method of switching the expression profile of the polypeptide of interest from the soluble form to the displayed form by inducing the expression of the suppressor tRNA.
  • FIG. IK is a graph that shows the expression profile of the antibodies as displayed proteins when the suppressor tRNA constructs are present in different expression vectors in HBV88.
  • the x axis indicates the dilution of supernatant containing the phage, and the y axis indicates the absorbance units measuring the product of the horseradish peroxidase activity.
  • the HRP here is conjugated to an anti-M13 monoclonal antibody.
  • FIG. 1L is a graph that shows the expression profile of the antibodies as displayed proteins when the suppressor tRNA constructs are present in different expression vectors in DH10BF' host cells.
  • the x axis indicates the dilution of supernatant containing the phage, and the y axis indicates the absorbance units measuring the product of the horseradish peroxidase activity .
  • the HRP here is conjugated to an anti-M 13 monoclonal antibody.
  • FIG. lM-1 and -2 are the Fast Protein Liquid Chromatography (FPLC) gel filtration evaluations of proteins produced from vectors, containing the variations in linkers between the BAP sequence and the sdAb/ His6 tag, and expressed in a conventional non-suppressor host strain HB2151.
  • the x axis indicates the elution volumes from the FPLC, and the y axis indicates the ultraviolet absorbance units for the protein contained in the fraction.
  • FIG. lN-1 and -2 are the FPLC evaluations of proteins produced from vectors, containing the variations in linkers between the BAP sequence and the sdAb/ His6 tag, and expressed in the conditional suppressor strain HBV88.
  • the x axis indicates the elution volumes from the FPLC, and the y axis indicates the absorbance units for the protein contained in the fraction.
  • FIG. lO is a picture of the polyacrylamide gel analysis of the proteins produced from vectors, containing the variations in linkers between the BAP sequence and the sdAb/ His6 tag. The upper half of the picture shows expression in the conventional non-suppressor host
  • FIG. lP-1, -2, -3, -4, -5 and -6 are the mass-spectrometry analysis of the peak tractions of the proteins produced from vectors, containing the variations in linkers between the BAP sequence and the sdAb/ His6 tag.
  • FIG. 1Q is a graph showing that the single species of sdAb produced within the host cell HBV88, used as both captor and tracer affinity agent, recognizes a polyvalent antigen, demonstrated by the highiy specific ELISA signals on positive control surrogate antigen.
  • the x axis indicates the volume of the shockate used as captor, and the y axis indicates the absorbance units measuring the product of the horseradish peroxidase activity. 10, 1 , 01 and 0.01 ⁇ , of the shockates were used either as captor (denoted on the x-axis) or as tracers (left to right bars in each vector set).
  • the HRP here is conjugated to neutravidin.
  • FIG. IR is a graph showing that the single species of sdAb produced within the host cell HBV88, used as both captor and tracer affinity agent, recognizes only the positive control surrogate antigen (FIG. 1Q) but not negative control surrogate antigen.
  • the x axis indicates the volume of the shockate used as captor, and the y axis refers to the absorbance units measuring the product of the horseradish peroxidase activity.
  • the HRP here is conjugated to neutravidin.
  • FIG. IS is a graph that shows the specificity of the rapid affinity agent pairing of 7 pairs of heterologous sdAb to the 7 serotypes of botulinum neurotoxin (BoNT).
  • the x axis indicates the seven different pairs individually challenged with each toxin serotype, and the y axis refers to the absorbance units measuring the product of the horseradish peroxidase activity.
  • the HRP here is conjugated to neutravidin.
  • FIG. IT is a graph that shows that both the standard display format (pecan21 ) armed with rapid affinity agent pairing ability (pecan 126) and the similarly armed rransdisplay system (pecanl33/134) can generate single domain antibodies specific for their desired target (BoNT serotype A) when screening from libraries of le +8 and l e : 9 respectively using a standard panning method.
  • the x axis indicates the antigens used to coat the plate, and the y axis refers to the absorbance units from measuring the product of the horseradish peroxidase activity.
  • the ITRP here is conjugated to an anti-M13 monoclonal antibody.
  • FIG. 1U is a dataset for monoclonal phage ELISA of clones from HBV88+ pecan 126 screened on plate coated with, botulinum neurotoxin A (BoNT A).
  • FIG. IV is the dataset for monoclonal phage ELISA of the same clones from HBV88+ pecanl 26 used above, screened on plate coated with ovalbumin.
  • FIG. IW is the dataset for positive clones identified from pecan 126/HBV88 heptaplex BoNT immune library, used as captor and tracer pairs in a checkerboard fashion, capable of recognizing i ug/mL BoNT serotype A in solution.
  • FIG. IX is the dataset for the negative control of FIG. IW. using the same clones from the pecanl 26/HBV88 heptaplex BoNT immune library on 1 ⁇ ⁇ , ovalbumin in solution.
  • FIG. 1Y is the amino acid sequence alignment of the sdAb clones selected using pecanl26 within HBV88 (126) and pecan 133 - ⁇ - pecan 134 within HB1251 ( 133) aligned with the original anti-BoNT A clones from conventional panning.
  • FIG. 2A is a schematic drawing of the rransdisplay system, wherein the display protein and the polypeptide of interest are encoded by separate expression cassettes in the same host cell.
  • FIG. 2B is an illustration of the nucleic acid compositions that may be used to establish the trans-display system.
  • FIG, 2C is a graph showing the EL1SA capture of sdAb (sdAb) or core streptavidin (STREP) displayed on the various coat proteins of M13 phage.
  • FIG, 2D is a graph showing the expression of streptavidin when using pSCUPER backbone vectors having the supE exchanged for supE- streptavidin-platform gene fusions.
  • FIG, 2E is a table demonstrating the checker-board optimization of arabinose (2000 to 2 ugrnL-l) and 1PTG concentrations (1000 to 1 ⁇ ) for transdisplay in HB2151 bearing supEVSS-sireptavidin platforms with pecan 133.
  • Signal here indicates measurement of the binding of phage-displayed sdAb to the positive Marburg antigen, measured indirectly as horseradish peroxidase activity.
  • the HRP here is conjugated to an anti-M13 monoclonal antibody.
  • FIG, 2F is a graph that shows that the transdisplay system can generate displayed antibodies against the Marburg virus nucleoprotein.
  • the x axis indicates the dilution of supernatant containing the phage, and the y axis indicates the absorbance units measuring the product of the horseradish peroxidase activity.
  • the HRP here is conjugated to an anti-M13 monoclonal antibody.
  • FIG. 2G is a graph showing that the transdisplay system can generate soluble biotinylated single domain antibodies and that these antibodies can function as captors and tracers in the rapid ligand pairing system to detect positive control antigen.
  • the x axis indicates the volume of sdAbs-eontaining shockate used, and the y axis refers to the absorbance units measuring the product of the horseradish peroxidase activity. 10, i, 01 and 0.01 ⁇ . of the shockates were used, either as captor (denoted on the x-axis) or as tracers (left to right bars in each vector set).
  • the HRP here is conjugated to neutravidin.
  • FIG. 2H is a graph showing that the transdisplay system can generate specific soluble biotinyiated single domain antibodies which do not recognize the negative control antigen.
  • FIG. 21 is the dataset of the monoclonal phage ELISA signals from 96 clones of pecanl33/134 immune heptaplex library on a plate coated with botulinum neurotoxin A ⁇ BoNT A).
  • FIG. 2J is the dataset for the monocional phage ELISA signals from the 96 clones of pecan 133/ 134 immune heptaplex library from FIG. 21 but on a plate coated with ovalbumin.
  • FIG. 2K is dataset obtained showing positive sdAb clones that were identified following selection and screening from pecan 133/134 retrofitted heptaplex BoNT immune library can be paired in a checkerboard fashion to recognize BoNT A in solution.
  • FIG. 2L is dataset obtained showing the lack of background signals when negative control antigen was used to identify sdAb clones.
  • FIG. 3A is a graph showing the enrichment of polyclonal phage specific for Ebolavirus Zaire (EBO) rather than Marburgvirus Musoke (MBG) through four rounds of panning a semi-synthetic sdAb library (ie. no bias to a given antigen/target) on purified Ebolavirus Zaire.
  • EBO Ebolavirus Zaire
  • MSG Marburgvirus Musoke
  • FIG. 3B is the amino acid sequence alignment of the anti-Ebolavirus Zaire clone isolated from transdisplay of the retrofitted single pot library, compared with the original sequence isolated via conventional panning.
  • FIG. 3C is a graph showing the single sdAb clone within HB2151 +pecanl 34 (EBC)Z C) produced as shockate for captor and phage as tracer to generate an Ebolavirus Zaire (EBO) specific assay, while MBGB captor and phage from HB2151+pecaii 134/133 served to confirm Marburgvirus (MBG) control was present.
  • EBC episomal sdAb clone within HB2151 +pecanl 34
  • EBO Ebolavirus Zaire
  • MBG Marburgvirus
  • HOUSTON4I30063 -14- Ebolvirus or Marburgvirus while y axis is absorbanee due to horseradish peroxidase colorimetric activity.
  • the peroxidase is conjugate to an anti-M 13 antibody.
  • FIG. 3D is a graph showing that at low virus numbers the system can struggle for sensitivity without phage amplification.
  • FIG, 4A is an exemplary illustration of the some vectors that may be assembled to select for improved soluble protein production.
  • a basal level of ⁇ -lactamase expression afforded by the sdAb fusion (pecan 148) provides an estimate of the basal expression level of a protein of interest must achieve in order to become expressed at equivalent soluble levels in pecanl48 HRP and ecan!48 SEP.
  • FIGS, 4B-D provide a demonstration of the method to select for enhanced soluble expression.
  • FIG, 4B is an illustration of the vector construct contained in the host cells in each of the five sections of the plate.
  • FIG. 4C is a picture that shows cells growing in the presence of arabinose 2000 at different concentrations of ampicillin and
  • FIG, 4D is a picture that shows cells growing in the absence of arabinose and at different concentrations of ampicillin.
  • a "nucleic acid” or a “nucleic acid composition” means any genetic element, capable of serving as a vehicle for genetic transfer, expression, or replication of a polynucleotide of choice in a cell.
  • the genetic element may also be functional in terms of expressing polypeptide's ⁇ in a cell-free system as is the case during in vitro transcription or translation reactions, and may be a circular replication competent motif or a linear expression cassette e.g. PGR product.
  • a nucleic acid composition may exist as a single polynucleotide or as two or more separate polynucleotides.
  • a nucleic acid composition may be a vector, a plasmid, phagemid, or a cosmid or it may be capable of stable integration into the host cell genome.
  • a nucleic acid composition may be a phage expression vector.
  • a nucleic acid composition may be capable of replication in eukaryotic cells or prokaryotic cells or both. It may be present as a single copy or in multiple copies inside a cell. Examples of useful nucleic acid compositions that can be modified for use in the present invention include, but are not limited to, the expression vectors - pecan series, pHEN series, pUC series, pAK series, pET series, and pBAD series.
  • the pecan series of vectors are proprietary vectors developed at the Texas Biomedical Research Institute.
  • An embodiment may include one or more genes inserted into an expression vector, in proper orientation and in proximity to a promoter such that under proper conditions, expression of the polynucleotide of choice can be directed in an appropriate host cell.
  • a nucleic acid composition may comprise at least one origin of replication and. may also comprise a gene for a marker by which it can be identified or selected, when inserted, into a host cell.
  • Useful markers are well known in the art and include for example, without limitations, markers that confer resistance to antibiotics, colorigenie or fluorogenic properties. The choice of a nucleic
  • HOUSTON4I30063 -16- acid composition will depend on the properties of the host cell and the desired properties of the polynucleotide of interest.
  • a “gene” refers to a nucleic acid sequence that comprises the coding sequences necessary for the production of a RNA or a polypeptide or their precursors.
  • a gene may include regulatory sequences preceding or following the coding sequence.
  • the term gene encompasses both the cDNA and genomic forms of the gene.
  • a “promoter” means a polynucleotide sequence in a nucleic acid composition that controls transcription of a nucleic acid sequence to which it is operably linked.
  • a promoter may include signals for RNA polymerase binding and transcription initiation.
  • the promoters used will be functional in the cell type of the host cell in which expression of the selected sequence is contemplated. Some promoters including constitutive, inducible and repressible promoters are well known in the art, and are available from a variety of different sources.
  • a promoter is usually located upstream of an expression cassette with the direction of transcription equivalent to the desired direction of translation of the polypeptide.
  • a promoter is typically a sequence or sequences with affinity for an RNA polymerase sufficient to induce binding that is required for transcriptional initiation.
  • a promoter may be repressible, inducible or constitutive.
  • a repressible promoter's rate of transcription decreases in response to a repressing agent.
  • a constitutive promoter's rate of transcription is not specifically regulated, though it can vary under the influence of general metabolic conditions.
  • An inducible promoter's rate of transcription increases in response to an inducing agent. Examples of such promoters with their inducing agents are, without limitations.
  • IPTG- inducibie lac promoter IPTG- inducibie lac promoter, tetracycline-inducible T7 promoter, arabinose-inducible promoters, salt- inducible promoters, temperature - inducible promoters.
  • a promoter may be able to drive a dynamic range of transcription levels by responding to the changes in the concentration of the inducer.
  • different promoters may be used to drive different expression profiles from the expression cassettes or nucleic acid compositions.
  • An "expression cassette” means a polynucleotide construct that contains coding- sequences for one or more proteins that may be operably linked to a promoter sequence.
  • An expression cassette may comprise other transcriptional regulatory sequences to direct proper transcription of the coding sequence into RNA.
  • An expression cassette may also comprise any of a variet of translation regulator)' sequences thai may be necessary or desired to direct proper translation of the RNA in the intended host cell.
  • the expression cassette is part of a nucleic acid composition and contains at least one gene that may be expressed by in vitro expression systems.
  • the expression cassette is part of a nucleic acid composition and contains at least one gene that may be expressed by the host cell.
  • the expression cassette may include other regulator)' ' sequences including, but are not limited to, an initiation codon for translation start, a termination codon for ending translation, an RNA splice site, a transcriptional termination site, and a polyadenyiation site.
  • the expression cassette may- contain the gene sequence for a protein of interest.
  • An expression cassette may contain coding sequences for a tag or a post-translational modification site.
  • An example of a post- translational modification site is the sequence coding for a biotin substrate site.
  • the expression cassette may contain a linker between the gene sequences for the polypeptide of interest and the post-translational modification site.
  • the expression cassette may also encode a protein that enables the post-translational modification of the protein.
  • the expression cassette may encode a biotinylating agent, like a biotin ligase that adds a biotin at the biotinylation substrate site of the polypeptide of interest.
  • a nucleotide composition or a sequence "encoding" a polypeptide or a gene means a nucleotide sequence that, when transcribed and/or expressed, results in the production of an
  • RNA polypeptide or protein.
  • the nucleotide sequence "encodes” that RNA or it encodes the amino acid sequence for that polypeptide or protein.
  • polypeptide of interest means an isolated or synthetic full length protein, an isolated or synthetic full length polypeptide, or an isolated or synthetic full length oligopeptide.
  • the terms polypeptide of interest or protein of interest may be used interchangeably.
  • a protein, polypeptide or oligopeptide has a minimum size of two amino acids.
  • Examples of recombinant polypeptides that can be used in the present invention include polypeptides derived from prokaryotic and eukaryotic organisms. Such organisms include phages, viruses, bacteria, fungi, plant or animals.
  • the polypeptide of interest can recognize or bind or otherwise interact with a molecule of interest.
  • the polypeptide of interest may be expressed as part of an expression cassette or by itself.
  • the coding sequence can be a native coding sequence for the polypeptide of interest or may be a coding sequence that has been selected, improved, or optimized for use in the host cell.
  • the polypeptide of interest may be obtained from an antibody library.
  • the polypeptide of interest may be obtained from a single chain antibody library, Fab library, sdAb library, knottin, ankyrin or other scaffold library.
  • the polypeptide of interest may be a single domain antibody.
  • the polypeptide of interest may begin with a signal sequence. Examples include, without limitations, leader sequences like pelB or DsbA.
  • the protein of interest may have a post- translational modification site. Post-transiationai modifications involve the addition of another moiety to an amino acid molecule of the polypeptide chain and include without limitations biotinyiation, giycosyiation or ubiquitination.
  • the post-translational modification site may be a biotin substrate site. This site allows a biotinylating agent to add a
  • biotin acceptor peptide contains a lysine that serves as the site of biotinyiation by E. coU biotin ligase though other naturally occurring or artificial biotin ligases may require different substrates.
  • the post-translational modification may also function as a tag.
  • a polypeptide of interest may function both as a captor and as a tracer.
  • a "biotin substrate site” means an amino acid sequence to which a biotin or a biotin analog can be added.
  • the biotin substrate site may be a biotin acceptor peptide.
  • the biotin substrate site may be fused to the protein of interest at any position.
  • the biotin substrate site may be C- or N-terminally fused in-frame to the protein of interest.
  • the biotin substrate site may be fused to the protein of interest at an internal position (e.g., a flexible internal loop).
  • the biotin substrate site may be designed to interfere minimally with the function or structure of the protein.
  • the biotinyiation of the protein of interest may be performed in a cell free environment or it may be performed in the context of a cell. Once biotinylated, the biotin molecule may also function as a tag for the detection, selection or purification of the polypeptide of interest.
  • a "biotinylating agent” or an “agent capable of biotinylating the polypeptide of interest” means a chemical or biological entity that can facilitate the attachment of a biotin or a biotin analog at a biotin substrate site.
  • biotinylating agent may be a biotin ligase.
  • the biotinylating agent may be expressed by a cell, either through an expression vector or may be stably integrated into the cell's genetic framework, or even supplemented into an in vitro reaction as protein, or incorporated synthetically during chemical synthesis of the affinity agent.
  • the biotinylating agent may be part of the expression cassette, carrying the protein of interest.
  • the cell may be a eukaryotie ceil or a prokaryotic ceil.
  • eukaiyotic cells include but are not limited to a mammalian cell, a Drosophila cell, a Xenopus cell, a
  • Providing an agent capable of biotinylating the polypeptide of interest includes all in vivo and in vitro means of facilitating the attachment of a biotin or a biotin analog at a biotin substrate site.
  • the biotinylating agent may be used with a protein of interest in a cell tree environment.
  • biotin protein ligase birA- Biotin holoenzyme synthetase
  • thai biotinylates the protein of interest at a biotin substrate site like Biotin Acceptor Peptide.
  • Deletions and low expression level formats of birA like GTG ⁇ birA may be better suited for the phage display where the acceptor peptide is fused to a phage coat protein.
  • a less modified birA using ATG initiation may be employed more effectively in the trans-display system, resulting in a better subsequent performance in the rapid pairing system.
  • a biotinylating agent is an example of a modification enabling protein.
  • Other modification enabling proteins can be devised to add post-translational modifications to the polypeptide of interest.
  • a “linker” means a polynucleotide or polypeptide sequence that is used in the expression cassette or fusion protein.
  • a linker may function to introduce cloning sites into the nucleotide sequence.
  • a linker may serve to as a tag for specific molecular interaction, like protein-protein interactions.
  • a linker may provide a flexible component or space- creating region between two protein domains.
  • a linker, separating the biotin substrate site and the polypeptide of interest, may facilitate the correct folding of the polypeptide of interest. Length of the linker is variable.
  • a linker can be a 15 -nucleotide sequence coding for the Gly-Gly-Gly-Gly- Ser sequence, which appears to improve the display of the affinity agent and also increases its subsequent production should purification be required.
  • a "display protein” means an amino acid sequence or a peptide or a protein that directs the presentation of a protein of interest.
  • a display protein may be a surface protein. Examples of surfaces for displaying the protein of interest may be, without limitation, the
  • a display protein may be a secretory protein, whereby the polypeptide of interest is now secreted by the host cell.
  • the display protein may be encoded by the same nucleic acid composition as the polypeptide of interest.
  • the display protein may be encoded on a different nucleic acid composition, or in the chromosome of the host cell
  • the display pro tern may be encoded in frame with an affinity agent.
  • a two nucleic acid construct system may be devised, where one nucleic acid construct encodes a polypeptide of interest with a post-translational modification site.
  • the second nucleic acid construct encodes a display protein and an affinity agent for that posttranslational modification. Interaction between the post-translational modification and the affinity agent leads to the sequestering of the polypeptide of interest and the display protein. The entire complex is presented by the display protein on its destined surface. By changing the na ture of the display protein, one can switch from bacterial surface display to phage surface display, without altering the construct that contains the polypeptide of interest.
  • Various surface proteins of a phage include the minor coat proteins g7p, g9p, g3p and g6p, and the major coat protein g8p.
  • Various surface proteins of E. coli include ice nucleation protein, ompA, and PAL.
  • Inner surface proteins anchor the polypeptide to the inner side of the outer membrane to face the periplasm, the periplasmic side of the inner membrane (e.g. APEX) or the inner side of the inner membrane.
  • the display protein may be associated with a ribosome or a mRNA or dsDNA display.
  • a "termination codon” or a stop codon means a nucleic acid sequence that signals the protein translation machinery in the ceil to stop farther translation of the sequence.
  • a termination codon may be one of the three natural codons— LJAA, UAG or UGA.
  • a termination codon may also be synthetic translation termination sequence.
  • a vector with a termination codon separating the polypeptide of choice and the display protein may be co-expressed with a suppressor tRNA.
  • the termination codon facilitates the switch in expression of the polypeptide of interest from a soluble form to a surface-displayed protein.
  • a termination codon may be present between the affinity agent and a display protein. This facilitates the production of independent affinity agents and also affinity agents fused to the display protein. When utilizing any termination codon as this molecular switch, it would be beneficial to ensure that the termination of transcription of the subsequent proteins in the expression cassette is not affected.
  • tRNA When a tRNA is said to recognize a codon, it refers to the process by which the anticodon of tRNA binds to the codon of the niRNA and delivers an appropriate amino acid to the ribosome for further polypeptide synthesis.
  • tag means a chemical or biological entity, which facilitates isolation, purification or detection of a polypeptide containing the entity.
  • a tag may be an amino acid sequence, or a nucleotide sequence that encodes an amino acid sequence.
  • Suitable tags include, but are not. limited to, HA peptide, polyhistidine peptides, streptavidin, and other antibody epitope binding sites.
  • Tags may be expressed as part of the polypeptide of interest as a fusion protein. Tags may be post- translational modifications, like biotin, that are added after the protein is expressed.
  • a tag may be a chemical entity.
  • a tag may be a fluorescent or a radioactive label that is attached to the polypeptide.
  • Detecting means direct or indirect measurement of the interaction between the molecule of interest and the protein of interest.
  • the measurement can be accomplished, by various methods known to one skilled in the art and includes, without limitation, absorbance, transmission, mass measurement, fluorescence intensity,
  • the interaction between the molecule of interest and the protein of interest is directly detected.
  • the interaction between the molecule of interest and the protein of interest is detected by detecting the tag.
  • the interaction between the molecule of interest and the protein of interest is detected by measuring the interaction of another affinity agent to the molecule of interest, or the protein of interest, or the complex between the molecule of interest and the protein of interest
  • affinity agent means a chemical or biological agent that interacts specifically with the molecule of interest, or the polypeptide of interest, or the tag on the polypeptide of interest.
  • affinity agents include a tamers, haptens, peptides, antibodies, antigens, streptavidin, or neutravidm.
  • Affinity agents may be synthetic equivalents of proteins or nucleic acids. The interaction between an affinity agent and. its target can include recognition, or binding, or alteration of physical or kinetic properties, or regulation of activity or location.
  • An affinity agent may be a polypeptide that recognizes and binds to a post-translational modification on the polypeptide of interest.
  • An affinity agent may be a bio tin affinity agent. Biotin affinity agents may include, without limitations, streptavidin, neutravidin or avidin proteins.
  • molecule of interest means any chemical or biological entity of interest for which an affinity agent is desired.
  • a molecule of interest can be without limitation a small molecule, peptide, hormone, antigen, antibody, deoxyribonucleic acid (DNA). ribonucleic acid (RNA), metabolites of the aforementioned materials and. other substances of either natural or synthetic origin.
  • Binding affinity means a selective or specific interaction between the molecule of interest and the affinity agent. Binding affinity may include formation of a bond between the
  • Binding affinity may also include noncovalent interactions such as hydrophobic bonds, salt links, hydrogen bonds and van der Waals forces.
  • an antigen and its specific antibody exhibit a specific binding affinity.
  • a "host cell” or “cell” means any cell of any organism that is selected, modified, transformed, grown or used or manipulated in any way for the production of a substance by the cell.
  • a host cell may be one that is manipulated to express a particular gene, a DNA or RNA sequence, a protein or an enzyme.
  • Host cells can further be used for screening or other assays to detect the presence of the particular biological product.
  • Host cells may be cultured in vitro or as one or more cells in a non-human animal (e.g., a transgenic animal or a transiently transfected animal).
  • Nucleic acid compositions according to the invention can be introduced into the target cells by various methods known to one skilled in the art.
  • the nucleic acid compositions can be expressed in cell- free systems. In another embodiment, the nucleic acid compositions can be expressed in synthetic formulations that support transcription and translation, for example liposomal formulations or encapsulated aqueous compartments.
  • an “antibody library” refers to a plurality of DNA or RNA molecules containing an open reading frame that encodes an antibody or fragment therof. It also includes a plurality of polypeptides expressed, from said DNA or RNA molecules. An antibody library may be available for further screening for specific properties,
  • a “single domain antibody” refers to a small functioning binding unit of an antibody.
  • a single domain antibody usually corresponds to the variable region of heavy chain only antibodies, though may refer to suitably engineered (e.g. "camelized”) heavy variable or light variable regions of conventional (heavy+light chain) antibodies, or variable regions exhibiting favorable solubility
  • a “suppressor tRJSIA” means a transfer RNA molecule that causes the incorporation of an amino acid in a polypeptide in a position corresponding to the termination codon in the mRNA being translated.
  • the expression of a product with a "native" carboxy end amino acid sequence occurs under non-suppressing conditions (i.e., when the suppressor tRNA is not expressed). While under suppressing conditions (i.e., when the suppressor tRNA is expressed), the protein translation machinery continues to translate the sequence subsequent to the termination codon, resulting in a fusion protein. Any tRNA suppressor, natural, synthetic or otherwise, and its matched stop codon pair may be used in the practice of the present invention.
  • the gene for the suppressor tRNA may be in the genome of the host cell
  • the gene for the tRNA suppressor may be located on the same nucleic acid composition or on a separate nucleic acid composition than the protein of interest.
  • the vector containing the suppressor tRNA gene may have an origin of replication selected so as to be compatible with the vector containing the expression cassette.
  • the genes may be expressed from the same or different promoters as compared, to the promoter used, to express the nucleic acid, encoding the protein of interest. Two or more different suppressor tRNA-termination codon pairs may be provided.
  • An "array” means an ordered arrangement of immobilized, molecules on a substrate.
  • a substrate may, without limitations, take the form of a particle, bead, gel, matrix, membrane, filter, chip, well, floweell or a trench.
  • the substrate may be coated with an affinity agent, or a polypeptide of interest or any other molecule of interest.
  • An array may contain a plurality of immobilized molecules.
  • An array may contain a plurality of
  • Nucleic acid compositions encoding polypeptides of interest are designed and formulated to obtain the desired level of transfer, replication and expression efficiency of the polypeptide of interest inside host cells. These nucleic acid compositions may be designed to function in cell-free expression systems or other in vitro systems. Generally, nucleic acid compositions are prepared to include a promoter sequence, and genes coding a selectable marker, a protein of interest, and a tag. A post-translational modification site may be cloned in-frame with the gene for the protein of interest, and this may also function as a tag. Standard recombinant DNA methods can be used to obtain desired nucleic acid compositions.
  • Cells can be prepared by starting with competent cells and introducing the exogenous nucleic acid compositions. Methods of introducing exogenous nucleic acids are known to one skilled in the art and include transformation, transfection, transduction or other physical or chemical or biological means. Cells containing the desired nucleic acid compositions are selectively propagated by using markers like antibiotic resistance or color expression systems. Host cells, containing the expression vector for the protein of interest, are generally selected and grown in appropriate media to desired cell population levels. Ceils are said to be incubated when they are grown or maintained in the desired, media at desired temperature, humidity, CO ? ,. G? and other conditions.
  • the cells When cells are incubated under “conditions sufficient for protein expression", the cells have all the nutrients, growth factors, and other materials and reagents required for expressing the polypeptides encoded by the nucleic acid compositions, such as the polypeptides of interest or affinity agents.
  • the inducing agent acts upon the promoter present on the nucleic acid compositions inside the cell. This drives the expression of the downstream expression cassette and thus producing the proteins encoded by the expression cassette.
  • IPTG isopropyl-bela-D- thiogalactopyranoside
  • the protein of interest may be identified, modified or purified by taking advantage of the distinct immunological, enzymatic or physical properties of the polypeptide. For example, and without limitation, if a protein or polypeptide has a unique enzymatic activity, an assay for that activity can be performed on the culture medium used by the host cells. Antibodies that recognize the polypeptide can be used to detect the protein or polypeptide in any immunological assay.
  • Cellular fraction means a collection of cells, including host cells which contain the polypeptide of interest, wherein such cells have been removed from their growth environment.
  • Cellular fraction for example, without limitations, include ceil lysates, where the cell membranes are disrupted by physiological or chemical means, and cell shockates.
  • Cellular fractions may include semi-purified or purified fractions of cellular extracts.
  • Chemical agents or biological agents may catalyze the post-translational modifications of the polypeptide of interest and this may happen in vivo or in vitro.
  • a post-translational modification like biotinylation may occur in a cell-free system, wherein an exogenous biotin ligase facilitates the addition of a biotin molecule at the biotin acceptor site of a polypeptide of interest. This in vitro
  • HOUSTON4I30063 -28- biotinylation involves exposing cellular fractions containing the polypeptide of interest with the biotin substrate site to the biotin ligase reaction mixture.
  • a biotin ligase may be encoded as part of the same nucleic acid composition as the polypeptide of interest. When expressed from the expression cassette after induction, this biotin ligase can add a biotin molecule at the biotin acceptor site of a polypeptide of interest.
  • a biotin ligase may be encoded by a different nucleic acid composition than the polypeptide of interest. Exogenous biotin may be provided to the host cells. Sufficient quantity of biotin may be present in the cell culture media to enable successful display and periplasmic production,
  • a host cell may contain several nucleic acid compositions.
  • the first vector comprises a first promoter and an expression cassette encoding a polypeptide of interest and a downstream display protein.
  • the gene sequences for the polypeptide of interest and. the display protein are separated by a termination codon.
  • the second vector in the same cell contains a second promoter and a suppressor tRNA gene that recognizes the termination codon in the first vector and suppresses the termination of protein synthesis.
  • the expression of the suppressor tRNA leads to the production of a proportion of the polypeptide of interest attached, to the display protein.
  • the polypeptide of interest may now be presented on a cellular or viral surface (or may be on ribosome if using an in vitro reaction). If the display protein contains the sequence for the phage coat, then the polypeptide of interest is presented on the phage coat, if the display protein moves to the periplasmic space, then the polypeptide of interest is presented in the periplasmic space.
  • HOUSTON4I30063 -29- agents one can change the expression profi le of the polypeptide of interest from a soluble form to a displayed form.
  • using inducible supE expression systems enabled single domain antibody populations to be propagated as phage for more panning from repertoires or expressed as soluble antibodies for screening within a single host strain.
  • the supE expression system provides for the expression of a mutant tRNA that will suppress the reading of the termination codon and permit the addition of an amino acid to the growing polypeptide chain.
  • the host cell comprises two nucleic acid compositions.
  • the first vector comprises a first promoter and an expression cassette encoding a polypeptide of interest and a post-translational modification site.
  • the second comprises a second promoter, and a second expression cassette encoding a downstream display pro tern and an affinity agent for the post-translational modification.
  • the post- translational modification site may be a biotin acceptor peptide and the corresponding affinity agent may be streptavidin. The interaction between the post-translational modification and the affinity agent leads to sequestering of the polypeptide of interest by the display protein.
  • the polypeptide of interest can be directed to any cellular surface of choice without re-engineering the polypeptide of interest into other vector constructs.
  • the nucleic acid composition comprising the polypeptide of interest may be used with display systems in different host cells. For example, by using yeast display proteins in a yeast host cell, the polypeptide of choice can be displayed on yeast ceil surfaces.
  • an inducible supE expression system was combined with the expression of streptavidin as a fusion protein with the gene 3 protein (g3p) of Ml 3 bacteriophage. This enabled a semi-synthetic single domain antibody library, which was mined for mti-Ebola virus antibodies, to be readily available for immunoassays without requiring recombinant protein purification.
  • the transdisplay system where the display protein with the affinity agent and the polypeptide of interest with the affinity tag are on separate nucleic acid compositions.
  • the nucleic acid composition in which the libraiy of polypeptides of interest is assembled, lacks the gene for the display protein and is therefore more efficiently transfected into host cells enabling larger repertoires to be assembled.
  • the 133 library size was approximately ten-fold larger.
  • a stronger promoter may be utilized for the expression of the polypeptide of interest. This will help avoid the toxicity or immunity to superinfection caused by overexpression of the display protein. Using a strong promoter for the polypeptide of interest will enable facile characterization and subsequent purification if required.
  • Certain polypeptides of interest may have C-terminal folding challenges. For example, and without limitation, a protein can have a buried C-terminal structure, so displaying such polypeptides as direct fusion products with the display protein may not be feasible. Transdisplay systems may alleviate this as the polypeptides of interest may be produced with only a tag and/or a flexible linker.
  • a multimeric affinity agent may be devised such that it binds to more than one polypeptide of interest with the affinity tag.
  • low affinity clones may be selected by virtue of avidity.
  • Different polypeptides of interest may be displayed simultaneously, by taking advantage of the multimeric nature of the affinity agent.
  • the invention provides for screening methods for identifying nucleic acid molecules, bacteriophages that may contain them, or the host cells that encode the polypeptides of interest against a molecule of interest.
  • the molecule of interest may be a chemical or a biological entity.
  • the molecule of interest may be a nucleic-acid-based or amino acid-based composition, or may be a naturally occurring or synthetic small molecule.
  • the molecule of interest may be a viral protein or a bacterial surface protein.
  • a polypeptide of interest may be a receptor with specific binding to a ligand of interest.
  • a polypeptide of interest may be an antibody that binds specifically to an antigen of interest.
  • host cells may contain polypeptides of interest that constitute one or more libraries and may encode variable domains of antibody light and heavy chains.
  • a library of clones displayed on phage is screened for specific properties against a molecule of interest. Specific properties may include greater binding affinities or greater stability. Clones, that test positive for the desired characteristics, are mobilized to an expression host, and expressed as discussed supra. In one embodiment, single domain antibody clones were developed against a particular molecule of interest. In order to develop diagnostic reagents, two non-competing polypeptides of interest have to be identified - one polypeptide to bind to the molecule of interest and another to recognize that bound polypeptide complex. The two polypeptides have to be unique in some fashion to allow r discrimination between the two in the signal amplification step.
  • One embodiment of the present invention is a method of utilizing the same polypeptide of interest, without any further modification, as both a captor and a tracer. For example, without limitations, an expression system was developed with
  • HOUSTON4I30063 -32- biotinylated captor antibodies A single biotin associated captor antibody was bound by a neutravidin coated platform, and the same style of biotinylated antibody was used as a tracer. The subsequent neutravidin-enzyme secondar conjugate onl recognized the free tracer biotin and not the occluded biotin on the captor. Both avidin and streptavidin have high affinities for biotin. This combined with, the almost quantitative concealment of the bound hapten ensured an almost irreversible captor platform with no background binding. This system provided independence from DNA sequencing or antibody purification.
  • This system may be used in any environment but has particular use in high containment laboratories since no agents need to be removed from the laboratory to develop diagnostics and therapeutic antibodies against highly infectious pathogens.
  • a simple pairing system was developed using microliter amounts of E. coli osmotic shockates containing site-specific biotinylated antibodies. These single domain antibodies performed as both the captor and tracer for polyvalent Marburg virus nucleoprotein.
  • pairs of single domain antibodies were developed to recognize seven botulinum (BoNT) serotypes, enabling specific recognition of the cognate serotype,
  • the array is designed to present affinity agents for the tag present on the polypeptide of interest.
  • the tag may be a post-translational modification on the protein, or another tag expressed in frame with the polypeptide of interest.
  • the polypeptide of interest may be a purified preparation or a semi-purified preparation.
  • the polypeptide of interest may be present in a purified or unpurified or semi-purified cellular fraction. This strategy permits the use of crude cellular fractions, without several rounds of purification. Cellular fractions include, without limitations, whole cell lysate, cell shockate, osmotic shockate or semi-purified, fractions thereof.
  • the polypeptide of interest that binds to its affinity agent on an array functions as the captor.
  • An array with the affinity agent bound to the captor is treated with the molecule of interest that is recognized by the captor. Then the cellular fraction containing the polypeptide of interest is again added and it now performs as the tracer.
  • the captor-bound molecule of interest binds to the tracer and this entire complex may be detected by using the tag on the tracer.
  • the tag may be detected using a detection system such as but not limited to fluorescent detection system, a luminescent detection system, a photographic film detection system, an enzyme detection system, or an optical detection system.
  • an array may be devised with biotin affinity agents, like neutravidin.
  • biotin affinity agents like neutravidin.
  • the neutravidin selectively binds the biotinylated polypeptides.
  • the molecule of interest is subsequently allowed to bind to the bound polypeptides.
  • the same preparation of biotinylated polypeptides used in the previous step is added to the bound molecule of interest.
  • the biotinylated polypeptides of interest now function as the tracer.
  • the biotin attached to the tracer polypeptides facilitates the detection of the bound complex.
  • the method may include saturation of the binding sites on the affinity agent after the first binding with the biotinylated polypeptides. Saturation may be achieved by blocking agents that contain biotin such as non-fat dried, milk. In one embodiment, the method further comprises removing unbound biotinylated proteins prior to detecting bound biotinylated proteins.
  • the method may include other steps known in the art to facilitate binding interactions, like washing between the various steps to eliminate non-specific binding.
  • Example 1 Example i .A
  • FIG. 1A An example of the molecular basis of the affinity agent pairing system is illustrated in FIG. 1A.
  • An affinity agent like neutravidin (cross) is passively absorbed on a surface (black line).
  • Crude osmotic shockate is applied to the system.
  • This shockate contains the protein of interest, like a biotinylated single domain antibody (pointed tab).
  • the biotin moiety (pointed end of tab) facilitates the binding of the antibody to the neutravidin.
  • This shockate functions as the source of both antigen capture and antigen tracing, with distinction by the secondary enzyme conjugate defined as the ratio of signal to noise. Unoccupied biotin binding sites on the neutravidin can be blocked with biotin (triangle) in milk.
  • the molecule of interest like an antigen (branch) is added and is captured by the immobilized neutravidin-sdAb combination. Crude osmotic shockate is again applied and the antigen captures more sdAbs. Now the biontinylated sdAbs function as the tracer. Neutravidin charged with horseradish peroxidase (cross with lightning bolts) is added which can only bind to that sdAb that has free biotin. The bound complex is detected through horseradish peroxidase detection system. This system produces a colored, fluorometric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified.
  • FIG. IB shows the initial expression constructs that were engineered to generate antibodies to serve as examples of affinity agents compatible with the rapid pairing system. These construct designs were used to balance in vivo sdAb biotinylation with phage display compatibility and effectiveness of capturing/tracing.
  • Pecan is a vector designation and basal vectors used to establish the rapid pairing system are all based on pecanl 14. This vector pecan 1 14 is a tac promoter-based high copy number vector driving expression of a His6 tagged sdAb to an amber codon and through to M13K07g3p (g3p) in the appropriate strain.
  • the BAP was inserted between the sdAb and His6 tag with flexible Gly4Ser linkers (G4S- BAP-G4S) to yield pecanl 22.
  • the birA from E. coli DH10B chromosome was inserted with a ribosome binding site downstream of g3p to provide extra biotinylating capacity in pecan 123. Since overexpression of birA maybe deleterious to phage display, constructs with various mutations were developed, like constructs with poorly initiated birA (pecan 124), the deletion of the DNA binding motif (pecan 125) and a poorly initiating version (pecanl26).
  • HOUSTON4I30063 [0105] The difference in behavior between the two assays can be rationalized by each bound phage is recognized by hundreds of copies of the anti-phage gSp HRP and amplifying any positive signal, while each bound sdAb will be recognized only by a single neutravidin HRP.
  • FIG. IE demonstrates that the antigens used are specifically capable of polyvalent binding with conventionally immobilized non-hiotinylated sdAb and sdAb-aikaline phosphatase tracers.
  • Recombinant Marburg and Ebola Zaire virus nucleoprotein mimics are polyvalent within crude cytosolic extracts of E. coli and therefore are acceptable surrogates for finding the affinity agents at biosafety level 2 (B8L-2).
  • Representatives of unique sdAb genes were mobilized to pecan22, a high level peripiasmic expression vector based on pMoPaclO but encoding only a C-terminal His6 tag instead of His-myc tag. Host E.
  • E. coli TunerTM strains with pRARE bearing tac promoter-based pecan42 driving cytosolic expressioil of tagless Marburg virus Musoke (MBG) or Ebolaviras Zaire Kikwit (EBO) nucleoprotein genes were generated. Clarified lysates of these E. coli were titrated over duplicate wells coated with 100 p,L of 100 nM unbiotinylated sdAb derived from the pecan22. EBOZ C was a clone selected on the Zaire virus and MBG C was a clone selected on the Marburg vims using standard display methods. Antigen capture was detected with 100 ⁇ -, of 100 nM sdAb-AP fusion and Pico- West chemiluminescent detection.
  • HOUSTON4I30063 -38- normalized for binding to directly immobilized antigen was examined among constructs Slaving different biotin ligase mutations in two different host cells.
  • XL- 1 Blue is a constitutive supE positive host, while HBV88 is an inducible host working in the display mode (FIGS. IF and 1G). Since the starting source for the rapid pairing method may be immune or nonimmune libraries, the vector producing high antigen binding responses like pecan 126 may be desired.
  • Pecan is the vector designation and this experiment involves pecanl 14 having no BAP, and pecanl 26- 132 having BAP tags but with varying linker regions.
  • the pecanl 26 hasG4S linkers on both sides of the BAP.
  • the pecanl 30 has no G4S linkers on either side of the B AP.
  • the pecanl 31 has the G4S only at the 5 'end of the BAP.
  • the pecan 132 has the G4S only at the 3'end. of the BAP, These constructs were examined in two different host cells - (FIGS. 1H and II).
  • the interactions in the host cell that lead to the phage display mode of the polypeptide of interest are shown above the dashed line.
  • Arabinose induction allows suppression of low level sdAb-amber-g3p translation and subsequent sdAb display.
  • soluble expression mode (below the dashed line)
  • the amber codon is not suppressed and full induction with IPTG generates large amounts of periplasmic sdAb.
  • the three vectors - K07 replicative form, pSCUPERV88 and pecan 126 - are represented as three large bold circles, and the supE V88 tRNA as a clover leaf.
  • the sdAb are represented as small circles and the g3p are represented as ovoids. Since pecan is a phagemid, it is prefereniiaily packaged during display over the K07 DNA.
  • the host strain of HB2151 + pSCUPERV88 is known as HBV88 and is used in this example. Consequently, HBV88 is highly effective at being able to express soluble sdAb when the supE V88 is switched, off, and. highly effective at being able to display sdAb on helper phage when the supE V88 is switched on (FIG. IS). HBV88 is capable of producing equivalent quantities and purities of sdAb as HB2151 following mid-scale expression and purification.
  • phage panning hosts are always supE+ve and so any progeny phage that are positive by monoclonal phage ELISA must be transferred to a non-supE host such as HB2151.
  • the transfer process leads to doubling of stored, clones and therefore, more prone to errors/mix- ups. This can hamper concise characterization.
  • HOUSTON4I30063 -40- protein can result in immunity to superinfection, and precludes the use of strong promoters for effective sdAh production and subsequent pairing. This apparently conflicting situation may be alleviated by engineering a plasmid that is compatible with pecan display vectors and conditionally expresses supE.
  • the vector pAR3 was initially chosen as a vehicle for supE, which is a pl5a origin chloramphenicol-resistant arabinose-inducible plasmid that is compatible with the colEl origin of the display phagemids. Though M13K07 helper phage also has a pi 5a origin, the different resistance genes between phage and plasmid should allow both to be selected for within the same cell.
  • a supE tRNA was cloned as a synthetic oligonucleotide bridge within the polvlinker of pAR3 (Perez-Perez and Gutierrez, 1995) to yield an arabinose-inducible supE cassette, pAR3supE.
  • pi 5a is quite a low copy number origin, and levels of suppression were insufficient in HB2151 bearing basal display vector pecan 114 when compared with XL-1 Blue (constitutively supE positive) (FIG. IK). Therefore, the gene dosage was elevated by fusing the chloramphenicol-resistant arabinose-inducible supE region of pAR3supE with the origin of an elevated copy number variant of pSClOl which enabled low but detectable display.
  • pSClOl is even lower than pi .5a, yet two high copy number mutants are known (Peterson and Phillips, 2008) with one exceeding p! 5a and so a chimera between pSClOl repA E ⁇ R and the Cm-Ara-supE portion of pAR3-supE was made using the splicing by overlap extension PGR approach to create pSCUPER.
  • the resulting plasmid began to show detectable levels of suppression of the amber codon within pecanl l 4 when both plasmids were maintained in the host HB2151.
  • HBV88 Increased suppression was afforded by replacing the wild, type supE gene with two individual point mutants known to increase suppression known as Su+2-88 and Su+2-89 (nicknamed herein as V88 and V89) (Bradley, et al, 1981).
  • the resulting strain of HB215 l+pSCUPER-V88 was denoted HBV88 and was first employed to
  • This strain HBV88 can be made electro competent by standard procedures to almost le+9 ⁇ u/ ⁇ g pUC and is therefore suitable for library generation.
  • Example IL 0113 The supE vehicles were compared in a strain other than HB2151 by conditionally transforming non-suppressor hosts into supE positive display hosts.
  • the highly electrocompetent strain DH10B was conjugated with XL 1 -Blue and progeny selected on streptomycin and tetracycline to make DHl OF'tet, a host suitable for phage display trials.
  • DHlOF'tet bearing pecanl l4 and the supE plasmids were superinfeeted with M13K07 and induced with 10 ⁇ IPTG.
  • Supernatants were analyzed, for capture by recombinant Marburgvirus NP (FIG. IL). Control signals on Ebolavirus NP were -0.010 to -0,002. This example demonstrates that the supE vehicles can perform in hosts other than HB2151.
  • FIG. lM-1 shows protein production from constructs - pecan73, pecan 114, and pecan 126 - in HB2151 strain.
  • FIG. 1M-2 shows protein production from constructs - pecan 130, pecanl31, and pecan 132 - in HB2151 strain.
  • FIG. 1M-1 shows protein production from constructs pecan73, pecan 114. and pecan 126 - in HBV88 strain.
  • FIG. 1M-2 shows protein production from constructs pecanl30, pecanl 31 , and pecan 132 - in HBV88 strain. Typical 400 mL shake flask cultures reliably yield approx.
  • the yield from pecan 126 and 132 is approx.75% of the yield of pecanl l4 (the basal vector without a BAP sequence), whereas pecan 130 and 131 are approx 30%.
  • pecan 126 consistently generated at least 30-100% more pure protein than pecanl30-132 (FIGS. IM and I N).
  • the differential production indicates the nature of flexibility around the BAP tag can influence yield. fOllS] Although the absence of the trailing Gly4Ser caused an approximate three-fold drop in soluble sdAb expression (FIGS.
  • FIG. 1Q and IR have two graphs that show the single species of sdAb produced within the host ceil HBV88 recognizing a polyvalent antigen, demonstrated by the highly specific ELISA signals on positive control surrogate antigen (FIG. 1Q) but not negative control surrogate antigen (FIG. IR).
  • FIG. 1Q positive control surrogate antigen
  • FIG. IR negative control surrogate antigen
  • This example shows the use of the present method in identifying pairs of antibodies and their complementary antigens.
  • One vector pecan! 32 was used within HBV88 to host a panel of sdAb that forms specific pairs capable of recognizing each of the 7 Bo T toxins to
  • HOUSTON4I30063 -44- confirm specific recognition of the cognate toxin serotype using just crude shockates (FIG. 18).
  • the sdAbs were produced in the pecan! 32 system and isolated as crude osmotic shocks.
  • Grade shockates of HBV88 were used as a source of captor and tracer, with a fixed 1 ⁇ / ⁇ , of toxin used.
  • Each pair was used as a captor-tracer combination and chailenged with a set concentration of each of the 7 BoNT serotypes to ensure specificity was retained, i.e., pair A bound only BoNTA, pair B bound only BoNT B etc.
  • specificity is retained on par with earlier findings on Luminex (the anti-D pair was raised against a C/D mosaic and hence shows reactivity with C and has also shown to cross-react with G).
  • FIG. ll is the graph that shows that both the standard display format (pecan21) armed with rapid ligand pairing ability (pecan 126) and the transdisplay system (pecanl33/134 similarly armed) can generate single domain antibodies specific for their desired target (BoNT serotype A) by screening from libraries of le+8 and le+9 respectively using a standard panning method.
  • FIG. IV is the representation of the same ELISA performed on ovalbumin. Positive clones, like that in well Al of FIG. 1U and FIG. IV, display a high signal to noise ratio, while negative clones like that in well A5 of FIG, 1U and FIG, IV. display a very low signal to noise ratio.
  • pecanl26 based display can serve as a productive resource for repertoires, enabling them to be rapidly deconvoluted. and resulting clones paired.
  • HOUSTON4I30063 -46- clone that is highly functional as a captor and a tracer, while clone Fl is an example of a poorly functional clone.
  • FIG. IX is the control assay on ⁇ g/mL ovalbumin. Negative control antigen ovalbumin 1 ⁇ g/mL used alongside the experimental pairing reveals no background signals after pairing sdAb clones isolated from the pecan 126/HBV88 mediated retrofit of a heptaplex BoNT immune library.
  • Clones from the conventional panning method start with the letter A indicating the anti-A serotype. Subsequent letter R and the number following it designate the round of isolation. Alpha-numeric designation following denotes historic ELISA plate well location. The final number designates specific clones, labeled from 1 through 18.
  • Clones selected from Example 1U start with the vector designation 126 followed by the well number from FIG. 1U.
  • Clones selected from Example 21 start with the vector designation 133 followed by the well number from FIG. 21.
  • Pecan 133 and pecan 134 share the lacZ' priming site and must be sequenced with AHX76. Clone D3 from the pecanl26 selection could not be fully sequenced, appearing to terminate before CDR3 and clone B8 from the pecanl33 selection terminated before the sdAb gene. Sequences were aligned with Multa!in.
  • HOUSTON4I30063 -47- Example 2 0126 As another example of the invention, we sought to reduce the size of the display phagemid to increase library sizes via improved transformation efficiencies by moving the platform gene (g3p) to the supE expression plasmid, while still retaining antibody pairing and single host capability. It should be possible for the g3p to display the sdAb in trans via capture of the hapten biotin, if it were afforded a streptavidin (strep) motif as shown in the schematic (FIG. 2A) alongside the phagemid-plasmid combination used (FIG. 2B).
  • strep streptavidin
  • FIG. 2A is a schematic of the transdisplay system.
  • the pecan! 34 vector a chloramphenicol resistant pSCl Ol high copy number origin mutant plasmid, expresses supEV88 which suppresses a sireptavidin core minigene fused to g3p platform.
  • the pecanl33 vector is an ampicillin resistant phagemid that expresses sdAb-BAP and birA only. Induction for soluble sdAb expression is the same as the HBV88 system, but for display the arabinose concentration is 100 fold lower to reduce toxicity from strep-g3p over expression.
  • the system was also developed to sequester the BAP within a phage displayed sireptavidin in trans by modifying the system in FIG. 1 J.
  • the displayed ligand e.g. sdAb
  • the displayed ligand is provided in trans from a pecan like phagemid. Display of the sdAb occurs since core sirepiavidin-g3p fusion binds to penplasmic sdAb-BAP and carries it out to the supernatant during the course of assembly (FIG. 2A).
  • the sdAb with BAP is expressed from pecan! 33 via IPTG induction and exported to the periplasm to become bound to the core-strep ⁇ g3p fusion and subsequently rransdisplayed on the assembled phage.
  • the core sireptavidin will assemble to a tetramer to be functional and may well capture multiple sdAb up to 4 maximally (each sireptavidin tetramer has 4 biotin binding sites).
  • the cell used is HB2151, which has no endogenous supE tRNA.
  • FIG. 2B is an illustration of the expression constructs that would function in a transdisplay mode and would be compatible with, the rapid pairing system. For example, a
  • the HOUSTON4I30063 -49- host cell may have two nucleic acid compositions.
  • the first nucleic acid composition comprises a polypeptide of interest (here it is a single domain antibody) in frame with a biotin substrate site, another affinity tag (e.g. His6) and a biotinylating agent.
  • the second nucleic acid composition comprises genes for a biotin affinity agent (e.g. streptavidin) and a display protein (e.g. g3p).
  • the genes for the biotin affinit agent and the display protein may be separated by a termination codon (marked as an asterisk on the second construct in FIG.
  • the gene for the suppressor tRNA that recognizes the termination codon may be present as part of the second nucleic acid composition (FIG. 2B) or it may be present independently on another nucleic acid composition.
  • the termination codon between the streptavidin gene and the display protein gene could be designed such that it permits read-through only about 20% of the time. This would ensure that at least three free streptavidin molecules are present to interact with one fused streptavidin-display protein molecule. This strategy may also eliminate the phage extrusion problems associated with self-assembly of the g3p protein.
  • the second nucleic acid composition in the host cell may be a dicistronic construct with at least two copies of the biotin affinity agent and no engineered termination codon between the biotin affinity agent and.
  • the display protein as there will be no requirement for a suppressor tRNA gene to facilitate the termination codon read-through, there will no rescue of host cells that contain termination codon errors in the polypeptide of interest.
  • This strategy may eliminate the need to repair error-prone PGR libraries, before using the constructs for high-level soluble expression.
  • the pecan 133 vector here is driven by the IPTG inducible tac promoter and pecan!34 by an arabinose promoter with the amber codon (*).
  • ELISA capture of sdAb (sdAb) or core streptavidin (STREP) displayed on the various coat proteins of Ml 3 within a pecanl l 4 backbone in XL-1 Blue were analyzed to choose the platform to proceed for transdisplay.
  • the coating antigen was either Marburg NP protein or an equivalent amount of the purified sdAb-BAP generated from pecan 126 (FIG. lO) to show that all platforms appeared to display enough streptavidin to be specifically bound by biotinylated sdAb.
  • Example 2C Constructs used in Example 2C were used to generate the transdisplay vectors. ELISA capture of pSCUPER backbone vectors having the supE exchanged for supE- streptavidin-platform gene fusions within XL-1 Blue initially was performed to verify that the streptavidin is still sufficiently displayed. All but g9p were capable of display (FIG. 21)).
  • HOUSTON4I30063 -51- HB2151 and combined transdisplav components (pecanl33+134 HB2151).
  • Signals on negative control Ebolavirus NP antigen ranged from 0.017 to -0.007.
  • FIG. 2F reveals that when in trans-displayed mode with 10 ⁇ IPTG and arabinose, the sdAb is displayed.
  • Positive control pecanl26 vector in either XL 1 -Blue or HBV88 was used to benchmark transdisplav.
  • Pecanl33, 134 and 133+134 were employed in HB2151 , such that 133 expresses just peripiasmic sdAb, 134 expresses supEV88-core-streptavidin-g3p and 133+134 expresses both and results in sdAb display, (Signals are specific for the Marburg nucleoprotein since simultaneous probing on Ebola virus NP yielded ranges of 0.017- 0.007).
  • transdisplay is that with a single library of sdAb (or other ligand) in pecanl33, resulting clones can be mobilized rapidly to any strep tavidin based display system without the need for subcloning.
  • Such systems may enable multicopy to single copy display- on phage by employing g8p rather than g3p as the streptavidin carrier to mimic high avidity low affinity to low avidity/high affinity selections.
  • it is not limited to phage display and may well also allow cell surface display via fusions of streptavidin to Ipp-omp.A or ICE nucleation protein, etc.
  • An advantage of the transdisplayed system will be the ability to display complex proteins that are not easily amenable to direct display, and that the final level of displayed protein will more accurately reflect the amounts occurring in the periplasm.
  • transdisplav vector pecan 133 is equivalent to the conventional display vector pecan 126 whether in HB2151 or HB2151 +pecan 134.
  • FIG. 2G reveals that when in soluble sdAb mode following 1 mM IPTG induction, the rapid ligand pairing system generated from pecan 133 is functional on par with previously
  • FIG. 2G shows the results when assayed with Marburg virus NP surrogate antigen and FJG. 2H with negative control Ebola virus NP antigen.
  • FIGS. 21 and 2J shows the examination of single monoclonal clones from the polyclonal response shown in FIG. IT for pecanl33/134.
  • the signals are relatively weak compared to 126 (since it is not as effective at display), there are clearly some specific clones here, like the clone in well A5.
  • FIG. 21 is the dataset for the monoclonal phage ELISA oil plate coated BoNT A
  • FIG. 2J is the dataset for the same ELIS A performed on ovalbumin. .
  • the library was made in a hygromycin resistant version of pecan 133 (pecan 164) to eliminate possible enrichment of existing sdAb in ampicillin display vectors and then selected on Ebolavirus Zaire (strain Kikwit 1995) at BSL- 4, Polyclonal phage ELISA showed enrichment of an Ebolavirus specific population (FIG. 3A). 22 of 22 clones picked from rounds 3 and 4 were sequences of a clone (EBOZ C) that we had isolated previously at a frequency of 1 1/26 (predicted amino acid sequences shown in FIG. 3B).
  • EBOZ C had never been individually mobilized to any pairing, transdisplay or hygromycin based vector and could only have arisen through enrichment from the retrofitted single-pot library.
  • the failure to isolate five other previously identified EBOZ sdAb clones could be due to their absence in the retrofit since it only matched the original library size and/or the log decrease in effectiveness of transdisplay observed in FIG. 2F,
  • the EBOZ C clone was directly employed via the dual use host HB2151+pecanl34 in a sdAb captor/phage tracer Filovirus titration (FIG. 3C) and a matching sdAb captor/sdAb tracer pairing (FIG. 3D).
  • FIG. 3A shows enrichment of polyclonal phage specific for Ebolavirus Zaire (EBO) rather than Marburgvirus Musoke (MBG) through four rounds of panning the semi-synthetic llama sdAb library Nomad#l within HygR pecan 133 (pecan 164) on Ebolavirus Zaire virus.
  • EBO Ebolavirus Zaire
  • MSG Marburgvirus Musoke
  • FIG. 3B show the predicted amino acid sequence of the anti-Ebolavirus Zaire clone isolated from transdisplay of the retrofitted single-pot library Nomad#l , compared with the original sequence isolated via conventional panning.
  • Example 3D 0145 Performed side by side with the phage tracer experiment in Example 3C on the very same dilutions of virus, we find using sdAb as both captor and tracer reveals that at these low virus numbers the system can struggle for sensitivity without phage amplification.
  • Another embodiment of the method would be screening for improved solubility variants.
  • an antibody clone maybe first screened to ensure binding has been retained.
  • By replacing the g3p display portion of the expression vector pecan 126 with mature ⁇ -lactamase one can select for mutant affinity agents with improved solubility by increasing ampicillin concentrations in HBV88 in display mode.
  • By switching to soluble mode one then has the direct means to screen any surviving clones for binding using a template similar to FIGS. 1Q and 1R and FIGS. 2G a 2H, aiming to generate high signals using increasingly smaller amounts of shockate as captor and tracer and it is very convenient to be able to switch from fused to unfused protein for enzymatic assay and characterization.
  • FIG. 4 A Vectors assembled for this task are shown in FIG. 4 A where a basal level of ⁇ -lactamase expression afforded by the sdAb fusion (pecan 148) gives one an estimate of the gain SBP and HRP must achieve in order to become useful genetic fusions in pecan 148 HRP and pecan 148 SBP.
  • Pecan 146 is a control vector with no ⁇ -lactamase resistance and pecan 150 is an example of the maximal level of ⁇ -lactamase resistance when no upstream motif is present, especially useful for the evolution of better sdAb solubility rather than HRP and SBP that would most likely be too much gain in one round of in vitro evolution.
  • These enzymes require heme to fold productively and the outer membrane of E. coll enables diffusion of this to the periplasmic compartment to enhance productive expression.
  • HOUSTON4I30063 -56- The reagents and methods described before for selecting polypeptides with greater binding affinity, can be used to select for improved solubility.
  • FIGS, 4B-4D phagemids were propagated in HBV88 overnight at 30°C and a loop streaked out onto 2xTY 2% glc plus Cm30 Hyg200.
  • FIG. 4B is an illustration describing the vector construct contained in the host cells in each of the five sections of the plates. 10 ⁇ IPTG is present in all plates.
  • FIG. 4C shows cells growing in the presence of arabmose 2000 at different concentrations of ampicillin and
  • FIG. 4D shows cells growing in the absence of arabinose at different concentrations of ampicillin. In both FIG. 4C and FIG.
  • the plates have different concentrations of ampicillin to measure how much soluble protein is produced.
  • the plate in the top right quartet has no ampicillin
  • the plate in the top left quartet has 25 fig/mL
  • of ampicillm the plate in the bottom right quartet has 100 ,ug/mL
  • the plate in the bottom left quartet has 400 ng/mL.
  • mutants on ara2000 and amp400 may be selected for variants that encode increased. Bla resistance.
  • HRP horse radish peroxidase
  • SBP soybean peroxidase
  • mutants on ara2000 and amp400 may be selected for variants that encode increased. Bla resistance.
  • soluble unfused proteins can be analysed for enzymatic activity (HRP and SBP for peroxidase activity ⁇ .
  • the sdAb serves as a calibration molecule. Fusion products like sdAb-HRP and sdAb-SBP may function as heat stable diagnostic reagents.
  • the single domain antibody may be evolved using a pecan 126/148 hybrid where g3p (on a HygR backbone) is replaced by Bla yet the BAP is retained to enable rapid screening of the ability to bind antigen as a sandwich.
  • Botulinum neurotoxins were handled in enhanced BSL-2 conditions while Filoviruses were handled in the full suit BSL-4 laboratory .
  • Non-biotinylating control vectors pecan45 was modified to pecan73 by exchange of the lac for toe promoter using technique of SQE-PCR (splicing by overlap extension by the polymerase chain reaction), and insertion of PCR amplified MBG B sdAb, specific for Marburgvirus nucleoprotein, from pecan2 lLgMBGB to delete the hinge and fuse framework four TVSS directly to the Noll site AAA reading frame to provide a basal hingeless pe1-sdAb-His6 expression cassette.
  • the full length g3p gene was PGR amplified from amberless pecan21 using primer NotHisamberTEV(5 ' - gaattc,gcg,gcc,gca,cat,cac,cat,cac,cat,cac,tag,ggt,ggc,ggt,gga,tct,gag,aat, ctt,tat,ttt,cag,ggc, gga,ggt,ggc,ggt,gct,gaa,act,gtt,gaa,agt,tgt-3') encoding His6 tag, amber codon plus a TEV protease cleavage site, and back primer g3pTAAHind3 (3'-tat,gac, gca,tta,ttc,ctc,aga,att,att,cgaaaaa-5'), and inserted into the N d and / indHI sites of pecan73 to provide
  • the g3p gene was sequenced using primers lacZ' (5 '-ctatgaccatgattacgaatttctag-3 '), AHX228 (5 '-cttatatcaaccetctcgacgge-3 '), AHX229 (5'-tcgtttgtgaatatcaaggcc-3 ') and AHX230 (5'-cattggtgacgtttccggcc-3').
  • Biotin acceptor peptide insertion Pecan 122 was made by replacing g3p from pecanl l 4 with a re-amplified product using front primer A'oiBAPHisg3p (5 '- ca,gcg,gcc,gca,gga,
  • BAP linker variants employed, a similar strategy but switching out the front primers with ones lacking the front (NotBAPG4SHisg3p), back (NotG4SBAPHisg3p) or both ( otBAPHisg3p noisyink) Gly4Ser coding sequences.
  • the g3p and linker sequences were verified as before.
  • the birA second cistron was PGR amplified from 1 ⁇ L of an overnight culture of E. coli DH10B. inserted into the HindlU site of pecan 122 and orientation mapped wrt internal Pstl and the framework 1 Pst of the sdAb.
  • the front primer for full-length birA was H3birAFOR (5 ' -aaaagcttaggaggacagctatg,aag,gat,aac,acc,gtg,cca-3 ' ) and for the deletion mutant34 was H3MaedaFOR.
  • HOUSTON4I30063 -60- and PacPSClOlFOR (5 '-cccaattaattatgattttttccccacgggag-3 ') with E93Rtop (5'- ggttaaaggctttcggattttccagtggac-3 ' ).
  • the products were then fused by SOE-PCR using the Pac primers for pull-through to create a 2.1 kbp high copy number pSCl Ol origin 12 segment.
  • the pAR3-supE was amplified using PacPAR3BAC (5 '-aaattaattaacetgaagteagccceatacg- 3 ') and PacPAR3FOR (5 ' -cccaatlaaitccgaataaatacctgtgacgg-3 ') to create the 2.0 kbp arabinose inducible supE segment bearing the chloramphenicol resistance gene.
  • sequencing was replaced by phenotypic screening in two ways. First, clones were benchmarked against the original high copy iiumber pSCl Ol mutants (generous gifts of Dr. Gregory Phillips) for approximate copy iiumber equivalence by agarose gel electrophoresis.
  • clones were mobilized to DHlOF'tet with pecanl 14 to screen for suppression in the presence of 2% glucose, 2000 .ugmL- arabinose and 1 niM IPTG following superinfection with M13K07 and kanamycin selection.
  • Suppression to enable MSG B sdAb display was monitored by phage ELISA with positives scored for signal on Marburgvirus nucleoprotein and not Ebolavirus nucleoprotein, and the highest signal to noise clone (#9) taken forward, pSCUPERV88 and pSCUPERV89 were made by replacing the wild type supE region between the Pstl and. ⁇ sites of pSCUPER with phosphorylated and.
  • bridges comprising either V88 top (5'-gtggggtatcgccaagcggtaaggcaecggatteta actecggcattccgaggttcgaatcctcgtaccccagccaa-3') plus V88 bottom (5'-agctttggetggggtacgag gattcgaaccteggaatgccggagttagaatccggtgcettaccgcttggcgataccecactgea-3') or V89 top (5'- gtggggtatcgccaagcggtaaggcaccggattctaaatccggcattecgaggttcgaatcetcgtaecccagecaa-3') plus V89 bottom.
  • the back primers were g7pTAAHwiO (3'- t,tag,cga,ccc,cca,gtt,cct.att.att.cgaaaa-5'), g8pTAAH «a'3 (3' ⁇ tgg,agc,itt,cgi,icg.att.att.cgaaaaa) and g9pTAAHwid3 (3'-gg,gca,aat,tac, ctt,tga,agg,agt.att.att.cgaaaaa-5').
  • the amplification products were used to replace g3p in pecanl 14 via Not!
  • pecanl 15 MBGB sdAb-gp9
  • 1 19 MBGB sdAb-gp7
  • pecan 120 MBGBsdAb-g8p.
  • Strep-display cassettes were PGR amplified using PstV881acZ (5 '-taactgeagtggggtat cgccaagcggtaaggcaccggattctaactccggcattccgaggttcgaatcctcgtaccccagccaatttattcaagacgcttaccttg taagtgcacccagtctatgaccatgattacgatct-3') which encodes the V88 supE mutant and intervening region between the two Gln2 tRNAs38 and the common back sequencing primer ⁇ 89 from respective strep-gp vectors and used to replace the resident supEv88 insert within pSCUPERV88 via Pstl and HmdllL
  • the starting constructs are the strep display vectors that fuse the strep minigene to one of the minor coat proteins, and the result are matching strep display vectors
  • the pecanl 33 vector was derived from pecanl26 by replacing the resident g3p and GTGAbirA cistron region via NotL and Hindill with a re- amplified GTGAbirA region made with Notl26Asc (5 '-tca,gcg,gcc,gca,gga,ggc,ggt, gga,tct,ggc,ctg,aac,gat,att,ttc,gaa,gct,cag,aaa,atc,gaa,tgg,cac,gag,ggc,ggt,gga,ggc,tct,cat,ca,cat
  • Hygfomvcin resistant sdAb transdisplay vector The hygromycin gene was a generous gift of Dr. Jim Sweigard and was isolated from pCB1004 by ligation in PGR using HygFRONTSwa (5 ' -aaggaaatttaa.atg,aaa,aag,cet,gaa,ctc,ac-3'), HygBACKSwa (3 ' - ca,ggc,tcc, cgt,ttc,ctt,atttaaatttatccgt-5 ' ), HygNcoDEL (5' ⁇ cag,ccg,gtc,gcg,gag,gcT,atg,gat,gcg,atc,gci,g-3') and HygNdeDEL (5'- ca,tgg,cgt,gat,ttc,atC,tgc,gcg,att,gct
  • HOUSTON4I30063 -63- into 100 ⁇ TFA The sample was bound to a C4 ZipTip (Millipore) by aspirating and dispensing into a second tube labeled "flo through" ⁇ 6 times, 20 ⁇ each. The C4 ZipTip was then washed with 100 ⁇ 0.2% TFA, and eluted into 50 ⁇ 50% acetonitrile, 0.5% acetic acid solution. The eluate was infused into a ThermoFinnigan Quantum triple quadrupole mass spectrometer at 0.6 ⁇ /min via a 250 ⁇ SGE glass syringe using the mass spectrometer's integrated syringe pump.
  • a New Objective egraFrit (IF360-75-50-N-5) was used inline as a pre-filter before the sample reached the New Objective Column Adapter (ADPC-IMS), which replaces the standard ESI probe of the ThermoFinnigan Ion Max source, and introduced into the mass spectrometer via a New Objective PicoFrit (PF36G-75-10-N-5) emitter.
  • the instrument was set in positive ion mode. Spray voltage was 2000V, and the heated capillary was set at 250°C.
  • Lysates were clarified by repeated eentrifugation and decanting (Allegra GPR, 5.75 krpm, 30 rain, 4°C), and finally filtration through a 0.22 ⁇ vacuum filter and stored in 2 mL aliquots at -80°C until required, ELISA titration of passively immobilized sdAb as captor and sdAb-AP as tracer in Tris buffered saline was used to verify that the antigens were a suitable crude surrogate for polyvalent viral NP with 10 ⁇ ih per well in MPBS determined to be our standard.
  • Nucleoproteins were produced for direct capture ELISA using a similar harvesting approach as applied to the C-terminally His tagged proteins in pecan42 to enable immobilized metal ion affinity chromatography (IMAC) purification of denatured monomer. Beadbeating to lyze the cells was carried out 100 mM TrisHCi pH 7.5, 100 mM NaCl, 5% glycerol, 6M guanidine HQ, 0,05% Tween-20, 10 mM ⁇ -mercapoethanol and 20 mM imidazole with protease inhibitor cocktail.
  • IMAC immobilized metal ion affinity chromatography
  • Clarified and filtered supernatant was applied with a superloop to a 1 mL nickel sepharose FF column and protein eluted via a 20 mM to 600 mM imidazole gradient. Peak fractions were silver stained and western blotted with anti- His HRP revealing expected 100 kDa band as the dominant species. These fractions were pooled and stored, in 100 ⁇ L ⁇ fractions at -20°C. Prior to use after thawing, the denatured preparation was microfuged to remove most of the aggregates that formed, with ELISA plate coating employing 100 ⁇ iL of a solution of ⁇ ⁇ , of the clarified nucleoprotem supernatant per mL PBS overnight at 4°C. Botulinum neurotoxins w r ere purchased from Metabioiogics (Madison, Wisconsin). 1 , ugmL-l in PBS was employed for panning, ELISA coats and pairing.
  • Ebolavirus Zaire strain Kikwit 19956 was amplified in 16 x 225 cm 5 flasks of Vero cells in DMEM/5% FBS/penicillin/streptomycm for 4 days. The 40 mL supernatants were collected, gently clarified by centrifugation (Allegra 6R, swing-out, 2.5 krpm, 5 min, 4°C) and stored at -80°C. Fresh media was added to the flasks and the amplification mixture was left for four more days.
  • Coats of virus for the first round of panning were 8 x 100 each containing approximately 4e+5 pfu.
  • Coats for rounds two through four were 8 x 25 ⁇ L ⁇ virus ⁇ 75 ,uL PBS each containing approximately le+5 pfu.
  • For polyclonal phage screening wells were coated with approximately 4e+3 pfu and for monoclonal phage screening wells were coated with approximately le+4 pfu.
  • Marburgvirus strain Musoke 1980 purified previously was used as the control virus.
  • Viruses were incubated in a final concentration of 0.1% Triton in MPBS for 10 min before applying to the ELISA plate to release the nucleocapsid.
  • 100 ⁇ of neutravidin (Pierce, Rockford, IL) at 1 ⁇ ⁇ in PBS was used to coat a 96 well high binding ELISA plate, either clear #3590 or white #3922 (Corning Costar, Tewksbury, MA ⁇ overnight at 4°C.
  • the plate was washed 3 times with PBS, and blocked by addition of 375 ⁇ , of PBS + 2% BSA + 0.1 % Tween-20 (PBSBT) for minimum of 1 h at room temperature.
  • the block was replaced by 100 ⁇ ih of fresh block containing the desired volume of shockate, typically 1 ⁇ .
  • the plate was washed 3x with 175 ⁇ , of PBS + 0.1 % Tween-20 and 2x with PBS. 100 ⁇ ih volumes of antigen were added in PBS + 2% non-fat dried Carnation milk (MPBS) and the plate shaken for 15-30 min. The plate was washed as before, the tracer (typically 1 ⁇ . of shockate) in 100 ⁇ iL volume of PBSBT was added and the plates shaken for 15-30 min. The plate was washed as before and 100 ⁇ .
  • Phage display and ELISA For conventional display and vector proving, 400 ⁇ ., of overnight cultures of phagemids in XL- lBlue/DH10-tetF' were used to inoculate 40 mL 2xTY 2% glucose plus appropriate antibiotics in 250 mL baffled flasks and shaken for approx. 2h at 37°C until an O.D. of 0.4-0.6 was reached.
  • M13K07 at a multiplicity of infection (moi) of 20 was mixed in, the culture left static for 30 min, and then shaken overnight at 30°C in the presence of 70 ⁇ ⁇ ⁇ 1 kanamycin and 10 ⁇ IPTG,
  • Display using HBV88 employed 10 ⁇ IPTG and 2000 ⁇ gmL ", L-arabinose while display in HB2151+pecanl34 utilized 10 ⁇ IPTG and 200 ⁇ gmL ", L-arabinose. 2 mL aliquots were
  • the destination vectors pecanl26, pecan 133 and pecan 164 were modified by replacement of the MBG B sdAb with a 2 kbp tetracycline resistance gene fragment from pecan21 via Ncol (partial)/ Noil to provide a convenient marker for scoring positive sdAb gene inserts and to enable SfillSfil cloning7 for the single-pot retrofit while immune libraries employed Sfil!Notl.
  • Each vector was grown in 12 x 3.5mL of terrific broth, 2% glucose and appropriate antibiotics and 12 x 2mL miniprepped for elution of approx. 2 x 0 g in 80 ⁇ iL EB which were then pooled. Vectors and.
  • inserts were digested by addition of 120 ⁇ 10 x React2 buffer, 120 ,uL 10 x BSA and 24 ⁇ iL of Sfil (20 ⁇ / ⁇ ) and left for 12- 18 h in a 50"C oven.
  • the immune library inserts and vectors were further digested by addition of 60 ⁇ xL React 3 and 24 ⁇ Noil (10 U/uL) for a iurther 12 h at 37°C.
  • Vector digests were dephosphorylated by addition of 120 ,uL !OxCIP buffer and 40 uL CIP (1 ⁇ / ⁇ ) and.
  • DNA was electrophoresed on 1% (for insert) or 0.5% (for vector) GTG TAE gels and the appropriate bands excised in a volume of about 2.5-3.5 mL for a scaled-up version of in agarose ligation. The gels were melted at 70°C then kept at 37°C
  • HOUSTON4I30063 -68- and both vector and insert were added and mixed in 400 ,uL aliquots into 6-8 tubes each containing 780 ,uL water, 200 ⁇ _ 10xT4 DNA ligase bu ffet (Proniega) and 20 uL ⁇ -Agarase ( ⁇ ⁇ / ⁇ ) which stayed liquid at room temperature (approx. 75F). 20 ⁇ _, of T4 DNA ligase ( ⁇ / ⁇ ) was added, the tubes mixed gently by inversion, covered in foil and left at slightly warmer than room temperature atop the hybridization overt for 1 8-24h.
  • the ligations were aliquoted to 24-32 x 500 p,L, each extracted with 450 .iL of phenol/chloroform (Invitrogen), and the 450 ⁇ , supematants precipitated by addition of sodium acetate/ethanol and left on the bench for 2h only to avoid excessive salt precipitation. Tubes were microfuged at 13.5 krpm for 1 5 min, supematants poured off, pellets briefly washed with 350 ⁇ 70% EtOH, recentrifuged for 5 min, aspirated and dried briefly in a tissue culture hood.
  • phenol/chloroform Invitrogen
  • Each pellet was resuspended in 42-32 ⁇ (depending on the number of ligations) and pooled to provide a combined 32 aliquots of 60 ⁇ for bulk electroporation which were aliquoted for storage at - 80 ' C until required.
  • HBV88 or HB2151+peca l34 were made electrocompetent using low temperature growth in low salt YENB media growth followed by extensive washing and reliably yielded transformation efficiencies in the mid l e+9 efu/ug ccc pUCI 9 range only some 2-4 fold less than home-made preparations of high efficiency strains such as DH10B or DHl OF'tet.
  • a streak on M9/chloramphenicol + thiamine minimal agar to select the F'-episome was rinsed into 400 mL of liquid. M9 equivalent and shaken overnight at 37°C.
  • HOUSTON4I30063 -69- washed in 15% glycerol and finally resuspended in 8.7 mL of 15% glycerol and aliquoted to 32 x 270 ⁇ , snap frozen in -80°C isopropanol and stored frozen until required.
  • Electroporations of the 60 ⁇ _. ligation and 270 ⁇ _. cell aliquots were performed using an eiectroporator (BioRad) at 2.5 kV with 2mm gap electroeuveties (Bulldog Bio Inc., Portsmouth. NH) that accommodated the combined volume. Following electroporation the cuvette contents were poured into 50 mL Falcon tubes followed by 3 cuvette washes of 2 mL of prewarmed (37°C) SOB 2% glucose with two electroporations worth pooled in each Falcon tube.
  • BioRad eiectroporator
  • 2mm gap electroeuveties Bulldog Bio Inc., Portsmouth. NH
  • Phagemids were pelleted (Allegra GPR, 3.75 krpm, 4x750 mL swing out, Ih, 4°C), drained and resuspended in a total volume of 16 mL PBS to which was added 16 mL of glycerol, and 16 x 2 mL aliquots stored at -80"C until required. *The remainder of the cell suspension was combined with an equal volume of ice cold 30 % glycerol in terrific broth and. aliquoted into 40-50 2 mL cryovials such that each aliquot could, seed another six flasks if required.
  • BoNT A the single round of panning was 20 washes each and for Ebolavirus Zaire it was 10, 20, 20, 30 each for rounds 1 through 4 respectively. 600 ⁇ ih of neutralized eluate was added to 10 mL of mid exponential phase HBV88 or HB 1251 +peca i34 as appropriate for 30 min before titrating an aliquot while the rest was gently pelleted, and plated on 15 cm diameter dishes. Overnight growth was followed by scraping large plates for glycerol stocking and liquid culture for M13K07 superinfection and display. Induction conditions are described above in phage display and ELISA. Polyclonal ELISAs utilized aliquots of the saved superinfected supematants while monoclonal ELISAs were derived from the titration plates.

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Abstract

La présente invention concerne la sélection et la production de protéines ou de peptides spécifiques avec des propriétés désirées. L'invention concerne des agents et des procédés d'identification de peptides ou de protéines sélectionnés avec des propriétés de liaison spécifiques ou des performances enzymatiques supérieures parmi un grand nombre de variants.
EP12819442.0A 2011-08-03 2012-08-03 Compositions d'acide nucléique, procédés et kits pour l'appariement rapide d'agents d'affinité Not-in-force EP2739733B1 (fr)

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